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Nervous Control of the Cornea

Carl F.Marfurt

Northwest Center for Medical Education, Indiana University School of Medicine, 3400 Broadway, Gary, IN 46408, USA

The cornea is the most richly innervated surface tissue in the body. It receives a dense sensory innervation from the trigeminal ganglion and a modest sympathetic innervation from the superior cervical ganglion. A sparse parasympathetic innervation has also been demonstrated in some species. Ocular fibres enter the cornea in various planes from the corneoscleral limbus and give rise to an elaborate, highly branched stromal nerve network. The nerves eventually enter the corneal epithelium and, after additional branching, terminate as free nerve endings. The nerves are not static structures, but demonstrate continuous elongation and terminal rearrangement under normal physiological conditions. The corneal innervation is neurochemically complex and individual corneal nerves may contain one or more of a dozen different neuropeptides and neurotransmitters, as well as a variety of neuroenzymes, cytoskeletal proteins, and cytoplasmic markers. Corneal afferent fibres serve important sensory, reflex, and trophic functions. The predominant, if not exclusive, sensory perception elicited by corneal stimulation is pain. Corneal unimodal and polymodal nociceptors are classified according to their relative abilities to transduce mechanical, thermal, or chemical stimuli. Stimulation of these nerves results in transmission of sensory information to the brain, initiation of the protective blink reflex, and the intraocular release of neuropeptides by axon reflex (neurogenic inflammation). Interruption of the ocular sensory innervation by disease or trauma produces a degenerative corneal condition known as neurotrophic keratitis. The pathogenesis of neurotrophic keratitis is multifactorial, but is due in part to the loss of trophic factors (possibly neuropeptides) supplied by corneal sensory nerves. Corneal sympathetic nerves also exert important trophic effects on the corneal epithelium; the latter nerves regulate corneal epithelial ion transport processes, cell proliferation and mitogenesis, and cell migration during corneal wound healing. Most mammalian corneas contain high concentrations of acetylcholine, choline acetyltransferase, acetylcholinesterase, and cholinergic receptors. Only a part of the cholinergic system is associated with the corneal nerves; most of it is

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associated with the corneal epithelial cells. The functions of the corneal cholinergic system are uncertain; however, the system has been implicated in the regulation of epithelial cell growth and proliferation, ion transport and sensory transduction mechanisms. Development of the corneal innervation begins in utero and is completed, depending on the species, either preor postnatally. It has been postulated that the developing nerves stimulate perinatal epithelial differentiation and the acquisition of corneal transparency.

KEY WORDS: corneal innervation; corneal pain; neurotrophic keratitis; sympathetic nerves

Correspondence: Tel: +1 219-980-6666; Fax: +1 219-980-6566; E-mail: cmarfurt@meded.iun.indiana.edu

INTRODUCTION

The cornea constitutes the most anterior surface of the eye. Histologically, it comprises a stratified squamous epithelium facing the ocular tear film, a dense connective tissue stroma, and a simple cuboidal endothelium facing the anterior chamber. Although the morphology of the cornea is relatively simple, its three layers are highly specialized for the refraction and transmission of light, and for the prevention of intraocular infection.

One of the most distinguishing features of the cornea is its rich nerve supply. The cornea receives a dense sensory innervation from the trigeminal ganglion and a modest sympathetic innervation from the superior cervical ganglion. In addition to the well known sensory and reflex functions of the corneal afferent fibres, corneal sensory and sympathetic nerves exert various “trophic” or nutritive effects on the cornea. These effects include the maintenance of epithelial cellular integrity, modulation of cell proliferation and mitosis, stimulation of ion transport, and regulation of wound healing after corneal injuries. In light of their numerous sensory, reflex and trophic functions, damage to the corneal innervation by trauma or disease may have serious visual consequences. The present chapter summarizes current knowledge of the origins, distribution patterns, ultrastructure, neurochemistry, electrophysiology, functions and development of the corneal innervation.

ORIGINS OF THE CORNEAL INNERVATION

SENSORY NERVES

Corneal sensory nerves originate predominantly, if not exclusively, from cell bodies in the medial, or ophthalmic, region of the ipsilateral trigeminal ganglion (Arvidson, 1977; Morgan, Nadelhaft and DeGroat, 1978; Marfurt, 1981; Marfurt and DelToro, 1987;

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Morgan, Janetta and DeGroat, 1987; Marfurt and Echtenkamp, 1988; tenTusscher, Klooster and Vrensen, 1988; Marfurt, Kingsley and Echtenkamp, 1989; Keller et al., 1991; Lavail, Welkin and Spencer, 1993). The results of retrograde nerve tracing studies suggest that 50–450 neurons innervate each cornea, the actual number depending on the species. Corneal-innervating neurons are predominantly small or medium in size, averaging 20–23 µm in diameter in rodents and 31–33 µm in larger mammals (Nishimori et al., 1986; Sugimoto, Takemura and Wakisaka, 1988; Marfurt, Kingsley and Echtenkamp, 1989; Keller et al., 1991). The relatively small sizes of the neuronal somata are consistent with the fact that these cells give origin to unmyelinated or finely myelinated fibres that conduct in the C-fibre or A-δ range (see below).

The sensory nerves reach the eye mainly, if not exclusively, via the nasociliary branch of the ophthalmic nerve. In humans, the nasociliary nerve gives rise to two or three long ciliary nerves which course directly to the posterior pole of the eye, and a communicating branch carrying sensory fibres to the ciliary ganglion (Figure 2.1). Approximately six short ciliary nerves, carrying a mixture of sensory and autonomic fibres, emerge from the anterior pole of the ciliary ganglion and, together with the long ciliary nerves, penetrate the posterior aspect of the globe in close proximity to the optic nerve.

Whether additional sensory fibres reach the cornea via the maxillary division of the trigeminal nerve has been debated. Clinically, interruption of the maxillary nerve in the orbital floor (Vonderahe, 1928) and transection of the maxillary portion of the trigeminal sensory root (Karvounis and Frangos, 1972) have been reported to produce impaired sensibility or total anesthesia in the lower half of the cornea. In contrast, other reports indicate that transection of the maxillary nerve at the foramen rotundum (Rowbotham, 1939) and traumatic injury of the infraorbital nerve (Norn, 1975) are without effect on corneal sensibility. The results of orbital dissections and nerve tracing studies in monkeys and cats have revealed a minor maxillary input to the cornea in these species (Ruskell, 1974; Morgan, Nadelhaft and DeGroat, 1978; Marfurt and Echtenkamp, 1988); however, transection of the cat maxillary nerve or rabbit infraorbital nerve revealed an absence of degenerating corneal fibres (Zander and Weddell, 1951b; Rodger, 1953). It may be concluded from these studies that the maxillary nerve in most cases provides little or no innervation to the cornea and, if present, is unlikely to provide meaningful preservation of corneal sensibility and trophic support following ophthalmic nerve lesions.

AUTONOMIC NERVES

In addition to a rich sensory innervation, the cornea receives a sparse to modest sympathetic innervation from the ipsilateral superior cervical ganglion (SCG) (Morgan, DeGroat and Janetta, 1987; Marfurt, Kingsley and Echtenkamp, 1989). In humans (Watson and Vijayan, 1995), sympathetic postganglionic fibres leave the SCG in the internal carotid nerve and ascend in the internal carotid plexus before entering the carotid canal in the petrous portion of the temporal bone. At the foramen lacerum, most of the ocular sympathetic fibres move anteriorly, away from the artery, and advance inferiorly and medially to the trigeminal ganglion to enter into a retro-orbital autonomic plexus with parasympathetic fibres from the pterygopalatine ganglion (Ruskell, 1970). The plexus forms a meshwork about the abducens, trochlear and ophthalmic nerves.

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Ultimately, most sympathetic nerves continue into the nasociliary nerve, and then into the long and short ciliary nerves, to reach the eye. Other sympathetic nerves pass forward in the walls of arteries, or independently, to reach the globe.

Although it is has long been assumed that the cornea is not innervated by parasympathetic nerves, recent observations have demonstrated their existence in some species. Application of the retrograde nerve tracing compound, horseradish peroxidase, to the central corneas of cats and rats consistently labels small numbers of parasympathetic neurons in the ipsilateral ciliary ganglion (Morgan, DeGroat and Janetta, 1987; Marfurt, Jones and Thrasher, 1998). In the rat, extirpation of the main ciliary ganglion causes degeneration of small numbers of corneal axons (Tervo et al., 1979). Conversely, surgical transection of rat ocular sensory and sympathetic nerves eliminates most, but clearly not all, corneal nerves (Marfurt, Jones and Thrasher, 1998).

NERVE DISTRIBUTION PATTERN

The anatomy of the mammalian corneal innervation has been the subject of intense investigation since the earliest description of these nerves over one hundred and sixty years ago. Regrettably, it is not possible in these limited pages to give recognition to the substantial contributions made by early investigators. For an excellent review of the literature before 1950 the interested reader is referred to the work of Zander and Weddell (1951a). In the latter paper, the authors published the results of a comprehensive study of the corneal innervation in rabbits, humans, and other vertebrates which has become the standard reference on the subject and on which much of the present day conception of the corneal innervation is based. These observations have since been confirmed and extended in a series of elegant studies in the rabbit (Robertson and Winkelmann, 1970; Tervo and Palkama, 1978b; Rózsa and Beuerman, 1982), cat (Chan-Ling, 1989) and human (Schimmelpfennig, 1982). The following description of corneal microscopic anatomy draws heavily on these and other accounts.

After piercing the sclera, ocular autonomic and sensory nerve fibres course towards the anterior eye segment in the so-called “suprachoroidal space”, located between the sclera and the choroid. Smaller numbers of fibres run forward within the sclera. As the nerve bundles run in the suprachoroidal space, they branch and exchange axons with one another such that by the time they reach the corneoscleral limbus each bundle contains a mixture of sensory, sympathetic and parasympathetic fibres. Near the corneoscleral limbus, the nerves destined to reach the cornea move anteriorly and separate from those supplying the anterior uvea.

THE LIMBAL AND STROMAL PLEXUSES (Figure 2.2)

Before entering the cornea, the nerves contribute fibres to a series of complex pericorneal (limbal) plexuses whose exact number and arrangement vary according to species. These plexuses, containing mixtures of sensory and autonomic nerves, form dense ring-like fibre networks that completely surround the peripheral cornea. The majority of the fibres supply a rich vasomotor innervation to the limbal blood vessels; however, others course

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within the limbal stroma apparently unrelated to vascular elements.

Sensory and autonomic nerves that supply the cornea pass through the limbus and enter the peripheral cornea in one of several planes. Most fibres penetrate at about midstromal level in a series of prominent, radially-directed nerve bundles. Approximately 70–80 fascicles, containing 900–1500 axons, enter the human cornea, while 20–40 major fascicles are typically seen in other mammals (Zander and Weddell, 1951a; Millodot, Lim and Ruskell, 1978; Chan-Ling, 1989). Other, smaller nerve fascicles enter the cornea in the episcleral and conjunctival planes to supply the superficial stroma and epithelium, respectively, of the peripheral cornea (Zander and Weddell, 1951a; Lim and Ruskell, 1978; Chan-Ling, 1989).

All corneal sensory nerves derive from finely myelinated or unmyelinated axons (Figure 2.3). In rabbits, more than 70% of the nerves are unmyelinated (Beuerman et al., 1983); the rest are finely myelinated (A-δ) axons that shed their myelin sheath within 1–2 mm after entering the cornea (Zander and Weddell, 1951a; Lim and Ruskell, 1978; Rózsa and Beuerman, 1982). In the human cornea, most unmyelinated stromal axons are about 0.5 µm in diameter; however, a few may be as large as 2.5 µm (Müller, Pels and Vrensen, 1996; Müller et al., 1997). Occasional myelinated axons are present in the central cornea in some species (Rodger, 1950; Whitear, 1960; Wakui and Sugiura, 1965).

Soon after entering the corneal stroma, the main bundles shed their perineurium and continue as flattened, ribbon-like structures sandwiched between the connective tissue lamellae. Adjacent nerve trunks branch, subdivide and rejoin with one another continuously in a series of irregular bifurcations or trifurcations to form a plexiform, multilayered network that distributes relatively uniformly throughout all four corneal quadrants (Ishida et al., 1984). Individual stromal axons may travel as much as threequarters of the way

across the cornea before ending (Zander and Weddell, 1951a, b). Not surprisingly, therefore, receptive fields of single corneal sensory axons may cover as much as 20–50% of the corneal surface (see below). The majority of the stromal nerves concentrate in the anterior one-third of the stroma, where they give rise to a dense subepithelial nerve plexus. The posterior stroma, on the other hand, is largely devoid of nerve fibres. A few workers have reported a sparse innervation of the corneal endothelium (Wolter, 1957; Leon-Feliu, Gómez-Ramos and Rodríguez-Echandia, 1978; ten Tusscher et al., 1989); however, the majority of investigators have been unable to substantiate these findings.

The question of whether some sensory fibres “terminate” in the stroma remains unsettled. Some authors have described the presence of thin nerve fibres that are confined entirely in their distribution to the stroma; the latter fibres end as a series of bead-like varicosities or are tipped by a single terminal expansion (Zander and Weddell, 1951a; Chan-Ling, 1989). Ultrastructurally, some stromal axons lack complete Schwann cell investments and lie in direct contact with the stromal extracellular environment (Tervo and Palkama, 1978a). Other fibres run in close vicinity to stromal keratocytes, occasionally invaginating the keratocyte cytoplasm (Müller, Pels and Vrensen, 1996). Many stromal axons display linear arrays of closely spaced varicosities containing small accumulations of clear and dense-cored vesicles, mitochondria, and other organelles (Matsuda, 1968; Hoyes and Barber, 1976, 1977; Lim and Ruskell, 1978; Tervo and Palkama, 1978a, b; Tervo et al., 1979). Morphological similarities between these

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varicosities and sensory free nerve endings in other tissues has prompted speculation that the stromal varicosities subserve sensory transduction functions, including, detection of lamellar shearing forces (Lim and Ruskell, 1978) and generation of pain associated with acute elevations of intraocular pressure (Zuazo, Ibañez and Belmonte, 1986).

Figure 2.2 Pattern of corneal sensory innervation in one quadrant of an adult rat eye. At the bottom of the figure, a dense, ring-like network of nerve fibres supplies the corneoscleral limbus. Nerves enter the corneal stroma either in prominent, radially-directly nerve bundles (large arrowheads) or in superficially placed branches off the limbal plexus (small arrowheads). The nerves then branch and anastomose repeatedly to give rise to a dense stromal plexus (large arrows) and large numbers of radially-oriented basal epithelial leashes (arrows). The nerves illustrated in this whole-mount preparation have been stained immunohistochemically with antibodies against calcitonin gene-related peptide. See text for details. (Reprinted from Jones and Marfurt, 1991, with permission of John Wiley and Sons, Inc.).

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Figure 2.3 Cross section of a small nerve bundle in the peripheral stroma of a human cornea. Several unmyelinated axons (asterisks) and two myelinated axons (My) are present. The nerve fibres are surrounded by cytoplasmic protrusions of perineural cells (arrowheads). Scale bar=2 µm. (Reprinted from Müller, Pels and Vrensen, 1996, with permission of Lippincott-Raven Publishers).

Intermingled with the stromal sensory nerve fibres are modest numbers of stromal sympathetic axons. The anatomy of the corneal sympathetic innervation has been investigated at the light microscopic level by histochemical fluorescence methods, immunohistochemistry, and nerve tracing techniques (Laties and Jacobowitz, 1964, 1966; Ehinger, 1966a, b, c; Ehinger and Sjöberg, 1971; Tervo and Palkama, 1976a, b; Klyce et al., 1986; Marfurt, 1988; Marfurt and Ellis, 1993). Sympathetic nerves, like corneal sensory nerves, are concentrated in the anterior one-third of the stroma (Figure 2.4); however, overall sympathetic innervation density varies considerably among species (Marfurt, Kingsley and Echtenkamp, 1989). In general, sympathetic nerves supply a modest innervation to the rabbit, cat and guinea pig cornea, and a relatively sparse innervation to the mouse, rat, hamster, and dog cornea (Ehinger, 1966c; Laties and Jacobowitz, 1966; Marfurt and Ellis, 1993). Sympathetic innervation of the adult primate cornea, including human cornea, had been previously reported to be absent or insignificant (Ehinger, 1966b, 1971; Laties and Jacobowitz, 1966; Sugiura and Yamaga,

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1968; Ehinger and Sjöberg, 1971; Toivanen et al., 1987); however, more recent studies of human corneas using antibodies against tyrosine hydroxylase suggest a more substantial sympathetic innervation than has been previously recognized (Ueda et al., 1989; Marfurt and Ellis, 1993).

Ultrastructurally, stromal sympathetic axons lack significant Schwann cell investments and may on occasion travel naked through the stromal matrix in direct contact with the extracellular environment (Tervo and Palkama, 1978a). The axons possess varicosities containing numerous small (300–500 Å), dense-cored vesicles thought to contain catecholamines; all of the axonal profiles with this morphology disappear after removal of the SCG (Tervo and Palkama, 1978a, b; Tervo et al., 1979).

EPITHELIAL NERVE FIBRES

Sensory nerves enter the overlying corneal epithelium either from the subepithelial plexus or, in the peripheral cornea, directly from the conjunctiva. As the nerves penetrate Bowman’s membrane, they shed their Schwann cell investments and continue into the epithelium as naked axon cylinders (Matsuda, 1968; Müller, Pels and Vrensen, 1996).

Intraepithelial sensory axons display a variety of morphologies; however, the two most common types of endings are complex basal epithelial “leashes”, and simple, vertically oriented branch-like structures (Rózsa and Beuerman, 1982; MacIver and Tanelian, 1993a, b). Basal epithelial leash formations are a unique form of nerve specialization found only in the cornea. Each leash consists of a family of 2–15, tightly packed thin axons that run approximately parallel to one another in the deep part of the basal epithelial layer, or between the basal epithelium and Bowman’s membrane (Figure 2.5). In most species, the leashes run in a predominantly radial (i.e., central) direction; however, in rabbits the leashes are oriented preferentially towards the nasal limbus (de Leeuw and Chan, 1989). Individual axons travel as far as several hundred microns in cats and rats (Chan-Ling, 1989; Jones and Marfurt, 1991) and up to 2 mm in humans (Schimmelpfennig, 1982). Occasional cross bridges interconnect adjacent axons. As the axons course horizontally through the basal epithelium, they give rise to an abundance of short, occasionally beaded, terminal axons which ascend vertically or obliquely into the more superficial epithelial layers before ending (Figure 2.6) (Rózsa and Beuerman, 1982; Ueda et al., 1989).

Numerous other epithelial nerves originate as single axons directly from the subepithelial plexus, penetrate Bowman’s membrane, and ascend vertically into the overlying epithelium (e.g., Rózsa and Beuerman, 1982; MacIver and Tanelian, 1993a, b). After a variable amount of additional branching, the fibres terminate in all layers of the epithelium (Figure 2.7).

Recently, MacIver and Tanelian (1993a, b) have described a third morphological type of intraepithelial axon in the rabbit cornea which outwardly resembles a basal epithelial leash formation, but which is located in the wing cell layer of the epithelium. This observation has not been confirmed by other workers.

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Figure 2.5 Leash formation in the basal epithelium of a rabbit cornea. The point at which the nerve penetrates the epithelium from the subepithelial plexus is indicated by the arrow. (Reprinted from Rózsa and Beuerman, 1982, with permission of Elsevier Science).

The innervation density of the corneal epithelium is probably the highest of any surface epithelium. It has been estimated that there are approximately 5000–8000 nerve terminals per square millimeter of rabbit central corneal epithelium (Rózsa and Beuerman, 1982), and one terminal per 20 square micrometers of human corneal epithelium (Schimmelpfennig, 1982). Most of the nerve terminals are located in the wing and basal cell layers (Rózsa and Beuerman 1982); however, terminals may also extend to within a few microns of the corneal surface (Rózsa and Beuerman, 1982; Burton, 1992; MacIver and Tanelian, 1993a, b). Innervation density (Rózsa and Beuerman, 1982; Chan-Ling, 1989) is closely associated with corneal sensitivity to mechanical stimulation (BobergAns, 1955; Cochet and Bonnet, 1960; Millodot and Larson, 1969; Draeger, 1984; ChanLing, 1989); both are maximal near the corneal apex and diminish progressively towards the corneoscleral limbus (Figure 2.8).

An undetermined percentage of intraepithelial nerve terminals are sympathetic. Intraepithelial sympathetic nerve fibres are relatively sparse in most species; however, a robust sympathetic innervation has been reported in the rabbit epithelium by horseradish peroxidase anterograde transport methods (Klyce et al., 1986).

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Figure 2.6 Innervation of the rabbit corneal epithelium. The innervation pattern shown here is based on camera lucida drawings and photomicrographs taken at successive focal levels. The size of the axon terminals relative to the dimensions of the epithelial cells is exaggerated for the sake of clarity. (Reprinted from Rózsa and Beuerman, 1982, with permission of Elsevier Science).

Corneal epithelial nerves are not static structures but continuously elongate and undergo morphological rearrangements under normal physiological conditions. The dynamics of the human corneal epithelial plexus have recently been studied in vivo by scanning slit confocal microscopy (Masters and Thaer, 1994; Auran et al., 1995). These workers have shown that stromal nerve trunks, subepithelial nerve fibres, and epithelial perforation points remain stable in position and topography over extended periods of time and therefore provide reliable reference points for tracking nerve movement in the overlying basal epithelial plexus. By monitoring positional changes in distinctive epithelial nerve features (e.g., branch points, axon kinks, etc.), Auran and coworkers (1995) concluded that basal epithelial nerves slide centripetally, in concert with the basal epithelial cells, at a rate of 10–20 µm per day. They further concluded that neurite growth occurs by the addition of new nerve material at the site of nerve entry into the epithelium, rather than at distal growth cones. Physiological rearrangement of epithelial nerve terminal morphology has also been demonstrated in living mice corneas stained with nontoxic fluorescent

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dyes (Harris and Purves, 1989). According to the latter authors, corneal epithelial nerves undergo both short-term, passive rearrangements in terminal configuration caused by the outward migration of differentiating epithelial cells, and long-term nerve-directed reorganization (Figure 2.9).

Figure 2.7 Examples of terminal arrangements of intraepithelial axons in the rabbit cornea. (Reprinted from Rózsa and Beuerman, 1982, with permission of Elsevier Science).

ULTRASTRUCTURAL OBSERVATIONS

The fine structure of corneal free nerve endings and their morphological relationships to corneal epithelial cells have been described by several workers (Whitear, 1960; Matsuda, 1968; Hoyes and Barber, 1976, 1977; Tervo and Palkama, 1978b; Tervo et al., 1979; Ueda et al., 1989; Müller, Pels and Vrensen, 1996; Müller et al., 1997). Intraepithelial varicosities, ranging from 0.2 to 1.8 µm in diameter, are found in several locations within the epithelium, including between the basal epithelial cells and Bowman’s membrane, between opposing cell membranes of adjacent epithelial cells, and invaginated within cytoplasmic infoldings of epithelial cells. The nerve endings are intimately apposed to the epithelial cell membrane, from which they are separated by a space of only 150–200 Å; however, membrane specializations and polarized accumulations of vesicles suggestive of “synaptic contacts” have not been described.

Two morphologically distinct types of sensory endings have been distinguished in some studies on the basis of organelle content (Matsuda, 1968; Hoyes and Barber, 1976, 1977; Ueda et al., 1989). One type contains numerous mitochondria, varying numbers of neurofilaments and microtubules, and occasional small, clear round vesicles. The second

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extirpation of the superior cervical ganglion and is therefore derived from corneal sympathetic nerves (Tervo and Palkama, 1978a, b; Tervo et al., 1979).

NEUROCHEMISTRY OF CORNEAL NERVES

Corneal nerves contain a variety of cytoskeletal and cytoplasmic proteins, neuroenzymes, neurotransmitters, and neuropeptides. Many of these substances are non-specific nerve markers with widespread distributions in ocular sensory and autonomic nerves, whereas others are exclusively or predominantly associated with corneal sensory, sympathetic, or parasympathetic nerves.

Figure 2.9 Remodeling of nerve terminals in the living mouse cornea as shown by staining the nerve endings with a nontoxic fluorescent dye. The anatomical constancy of the stromal fibre bundles (stipple) allows the same region of the corneal surface to be examined at different times. After one month, nerve terminal rearrangement is so extensive that neither the location of terminal clusters nor their detailed structure bear any resemblance to the initial pattern of endings observed within the same corneal region. (Reprinted from Harris and Purves, 1989, with permission of the Society for Neuroscience).

GENERAL MARKERS

Substantial numbers of corneal sensory and autonomic nerves contain the cytoplasmic markers, protein gene product 9.5 (PGP 9.5) (Marfurt, Ellis and Jones, 1993; Marfurt, Jones and Thrasher, 1998) and peripherin (Marfurt, Jones and Thrasher, 1998) as well as

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the neuroenzymes, neuron specific enolase (NSE) (Ueda et al., 1989) and acetylcholinesterase (AChE) (Laties and Jacobowitz, 1964, 1966; Petersen, Keat-Jin and Donn, 1965; Robertson and Winkelmann, 1970; Howard, Zadunaisky and Dunn, 1975; Tervo, 1976, 1977; Tervo and Palkama, 1978b). The presence of the latter enzyme suggested to early investigators a parasympathetic origin for these nerves; however, it is now known that AChE is a nonspecific marker that is expressed in large percentages of sensory and autonomic neurons. In the cornea, most AChE-positive nerves disappear after transection of the ophthalmic nerve and are therefore derived from the trigeminal ganglion (Tervo, 1976).

Many corneal nerves also stain immunohistochemically for growth associated protein43 (GAP-43) (Martin and Bazan, 1992). GAP-43 is a neuron-specific protein found in growth cones during development or regeneration of axons. The presence of this protein in mature corneal nerves is thought to be associated with the continuous process of intraepithelial axon elongation and terminal remodeling that occurs under normal physiological conditions (Harris and Purves, 1989; Auran et al., 1995) and during corneal wound healing (Lin and Bazan, 1995).

SENSORY NERVES

Corneal sensory nerves contain a variety of neuropeptides. Two peptides in particular, SP and CGRP (Figure 2.11), are found in large percentages of corneal sensory nerves (Miller et al., 1981; Tervo et al., 1981a, b; 1982a, b; 1983; Stone, Laties and Brecha, 1982; Ehinger et al., 1983; Sasaoka et al., 1984; Stone and Kuwayama, 1985; Stone et al., 1986; Kuwayama and Stone, 1987; Stone and McGlinn, 1988; Harti, Sharkey and Pierau, 1989; Silverman and Kruger, 1989; Uusitalo, Krootila and Palkama, 1989; Jones and Marfurt, 1991, 1998). The majority of nerves that contain CGRP also contain SP (Kuwayama and Stone, 1987) and electron microscopic investigations of rat corneal nerves (Beckers et al., 1992, 1993) suggest that the peptides may colocalize in the same vesicles (Gulbenkian et al., 1986; Merighi et al., 1988; Kummer, Fischer and Heym, 1989). Comparative radioimmunoassay studies reveal that corneal SP levels in small mammals are approximately 2–4 times higher than those in larger mammals (Gamse et al., 1981; Unger et al., 1981; Bucsics, Holzer and Lembeck, 1983; Elbadri et al., 1991).

A robust population of corneal galanin-immunoreactive (-IR) nerves, rivaling in density those of the SP and CGRP fibre populations, innervates the rat eye (Jones and Marfurt, 1998). Smaller numbers of galanin-IR nerves have also been demonstrated in porcine corneas (Stone, McGlinn and Kuwayama, 1988). Approximately 95% of rat galanin-IR corneal fibres disappear after ophthalmomaxillary nerve transection, thus demonstrating their sensory origin (Jones and Marfurt, 1998). In the rodent eye, galaninIR fibres apparently constitute a separate population of peptidergic fibres distinct from those that contain SP and CGRP because the former fibres (unlike ocular SP-IR and CGRP-IR nerves) are unaffected by neonatal capsaicin administration (Stromberg et al., 1987).

Pituitary adenylate cyclase-activating peptide (PACAP), a neuropeptide with strong structural homology to vasoactive intestinal polypeptide (VIP), has been demonstrated in corneal sensory nerves in rats and rabbits (Moller et al., 1993; Wang, Alm and Håkanson,

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1995), but not in cats (Elsås, Uddman and Sundler, 1996). PACAP colocalizes extensively with CGRP and SP in ocular sensory nerves and in trigeminal ganglion neurons.

Many corneal sensory nerves contain the enzyme, fluoride-resistant acid phosphatase (FRAP) (Szönyi, Knyihar and Csillik, 1979; Silverman and Kruger, 1988a, b). FRAP-IR nerves also express a surface oligosaccharide which binds the isolectin I-B4 of Griffonia simplicifolia (GSA I-B4), but they do not contain CGRP (Silverman and Kruger, 1988b). The function of FRAP in corneal nerves is unclear; however, it has been hypothesized that the enzyme may play a role in transmitter metabolism (Szönyi, Knyihar and Csillik, 1979). Finally, some corneal sensory axons in rats and mice, and possibly in humans, contain tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis (Ueda et al., 1989; Marfurt and Ellis, 1993).

AUTONOMIC NERVES

Most, if not all, corneal sympathetic nerves contain the classical neurotransmitter, noradrenaline (e.g., Laties and Jacobowitz, 1964, 1966; Ehinger, 1966a, b, c) and the catecholamine synthesizing enzyme, tyrosine hydroxylase (Ueda et al., 1989; Marfurt and Ellis, 1993). Many corneal sympathetic fibres also contain serotonin (Uusitalo et al., 1982; Osborne, 1983; Palkama, Uusitalo and Lehtosalo, 1984; Osborne and Tobin, 1987) and neuropeptide Y (NPY) (Stone, 1986; Stone, Laties and Emson, 1986; Jones and Marfurt, 1998) (Figure 2.12).

As noted earlier, a modest parasympathetic innervation of the cornea has been demonstrated in cats and rats by horseradish peroxidase nerve tracing methods and/or selective ocular denervations coupled with immunohistochemistry. In the rat, corneal parasympathetic nerves express VIP, met-enkephalin, NPY, and galanin (Figure 2.12) (Jones and Marfurt, 1998). A few (presumably parasympathetic) rat corneal fibres also contain the enzyme, nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase (Yamamoto et al., 1993). Measurable quantities of VIP (Unger et al., 1981; Elbadri et al., 1991) and the opioid-like peptides met-enkephalin, β-endorphin, and α-melanocyte- stimulating hormone (α-MSH) (Tinsley et al., 1989) have also been reported in corneas from larger mammals.

OTHER MARKERS

Several other peptides have been demonstrated by immunohistochemistry or radioimmunoassay in corneal nerve fibres, including cholecystokinin (Palkama, Uusitalo and Lehtosalo, 1986), brain natriuretic peptide (Yamamoto, McGlinn and Stone, 1991), vasopressin (Too et al., 1989) and neurotensin (Tinsley et al., 1989). At present, the origins, densities, and functions of these peptidergic nerve populations remain unknown.

The preceding account only hints at the complex neurochemistry of the corneal innervation. The percentage of corneal nerves that contain particular neurochemicals, and the extent to which individual neurochemicals colocalize and are co-released by subpopulations of corneal nerves, remain largely unknown. The impressive number of neuropeptides identified within corneal nerves, and recent demonstrations of peptide

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colocalization, suggest complex physiological interactions (Stone, Kuwayama and Laties, 1987). Until it is determined how particular neurochemicals correlate with specific sensory and “effector” functions of corneal nerves, the importance of each of these markers in corneal physiology is difficult to evaluate. It would be of great interest to learn, for example, if neurochemically distinct subpopulations of corneal nerve fibres (e.g., FRAP-IR and CGRP-IR) represent functionally distinct fibre populations for parallel processing of corneal nociceptive information, or if they subserve separate and distinct sensory, trophic, and inflammatory functions.

CORNEAL SENSORY INNERVATION: FUNCTIONAL

CONSIDERATIONS

SENSORY MODALITIES

It is generally stated that only nociceptive sensations are evoked by naturally occurring corneal stimulation. However, the question of whether “pure” mechanical or “pure” thermal sensations (i.e., tactile or thermal sensations uncontaminated with any nociceptive component) can be perceived in human psychophysical experience remains unresolved. The issue has fostered a century-long, spirited debate in which “Workers seem to have arrayed themselves as contestants, those who subscribe to von Frey’s theory [that nociception is the sole modality transduced by corneal receptors] on the one hand, those who do not on the other” (Lele and Weddell, 1956). The question has been difficult to answer due largely to methodological difficulties, including, high levels of test subject apprehension (Maurice, 1984).

According to some workers, mechanical (von Frey, 1894) and thermal (Nafe and Wagoner, 1937; Kenshalo, 1960; Beuerman and Tanelian, 1979) stimulation of the human cornea (carefully controlled to prevent spread to adjacent tissues) evokes only perceptions of pain. Identical stimuli perceived as painful when applied to the cornea are sensed as tactile, warm or cold when applied to adjacent regions of the conjunctiva, eyelid, or facial skin. Electrophysiological investigations in cats and rabbits suggest that some corneal afferent fibres are relatively modality-specific and that distinct fibre populations are activated preferentially by mechanical, thermal, or chemical stimulation (see below). It has been claimed that this modality-specificity is lost during subsequent central nervous system processing (Tanelian and Beuerman, 1984; MacIver and Tanelian, 1993a, b) and that all mechanical and thermal corneal stimuli are perceived as irritating or painful.

In contrast to the above point of view, other workers contend that graded punctate stimuli applied to the corneal surface with a smooth nylon monofilament under highly controlled testing conditions evoke pure (often liminal) sensations of touch (Lele and Weddell, 1956; Millodot, 1968; Draeger, 1984). As the pressure exerted by the nylon thread increases to many times the corneal touch threshold (Millodot, 1968), or if the stimulus remains in position for more than one second (Draeger, 1984), the sensation changes to pain. Other evidence in support of the existence of corneal tactile sensibility comes from observations in patients suffering from trigeminal neuralgia. Surgical

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interruption of the corneal central nociceptive pathways in these patients (i.e., trigeminal tractotomy) changes their perception of corneal stimulation from pain to touch (Rowbotham, 1939; Grant, Groff and Lewy, 1940; Falconer, 1949).

DETERMINANTS OF CORNEAL SENSITIVITY

Corneal sensitivity to mechanical stimulation is highest in the central cornea and decreases progressively through the peripheral cornea, limbus, and conjunctiva (BobergAns, 1955; Cochet and Bonnet, 1960; Millodot and Larson, 1969; Draeger, 1984; ChanLing, 1989). Sensitivity is adversely affected by several factors, including age, extended contact lens wear, eyelid closure during sleep, iris color, menstruation and pregnancy, environmental and atmospheric factors, and most ocular and many non-ocular diseases. Several in depth reviews are available on this subject (Draeger, 1984; Millodot, 1984; Martin and Safran, 1988).

ELECTROPHYSIOLOGY

Receptive fields

Receptive fields of individual corneal afferent fibres are generally round, oval, or wedgeshaped in outline, variable in size, and exhibit considerable overlap. A typical

mechanoreceptive field in the rabbit or cat cornea extends over 5–20% (10–20 mm2) of the corneal surface, although individual receptive fields may be as small as 1 square

millimeter or as much as 50–100 mm2 (Tower, 1940; Lele and Weddell, 1959; Mark and Maurice, 1977; Giraldez, Geijo and Belmonte, 1979; Belmonte and Giraldez, 1981; Tanelian and Beuerman, 1984; Belmonte et al., 1991). The largest receptive fields cover over 40–50% of the corneal surface. Receptive fields may also extend several millimeters beyond the cornea onto the adjacent limbus and bulbar conjunctiva. The large size and extensive overlap among adjacent receptive fields, coupled with convergence of sensory inputs onto common second order neurons in the trigeminal brainstem nuclear complex, explains why sensations arising from corneal stimulation are poorly localized (Lele and Weddell, 1956; Beuerman and Tanelian, 1979; Draeger, 1984).

Conduction velocities

All corneal sensory nerves originate from unmyelinated or finely myelinated fibres conducting in the C-fibre (0.25–2.0 m/s) or A-δ (greater than 2.0 m/s) range (Giraldez, Geijo and Belmonte, 1979; Belmonte and Giraldez, 1981; Belmonte et al., 1991; Gallar et al., 1993; MacIver and Tanelian, 1993a, b). The conduction velocities of individual nerves decrease significantly in the proximodistal direction (Belmonte et al., 1991; MacIver and Tanelian, 1993a, b) as the result of progressive intracorneal tapering of the axon and, in the case of A-δ fibres, shedding of the myelin sheath at the corneoscleral limbus.

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Modality specificity

The electrophysiological properties of corneal afferent nerve fibres in cats and rabbits have been well characterized (Figure 2.13) (see Belmonte and Gallar, 1996; Belmonte, Garcia-Hirschfeld and Gallar, 1997, for recent reviews). In general, corneal afferent neurons are either unimodal (responding to only a single modality of stimulation) or bimodal/ polymodal (responding to more than one modality of stimulation).

Three types of corneal unimodal units, mechanosensitive, cold sensitive, and chemosensitive, have been described by various authors. Corneal mechanosensitive fibres are fast conducting, rapidly adapting, A-δ afferents that respond exclusively to mechanical stimulation (indentation) of the corneal surface. These fibres exhibit velocity and force sensitivity and are especially sensitive to moving stimuli on the corneal surface (Lele and Weddell, 1959; Belmonte and Giraldez, 1981; Tanelian and Beuerman, 1984; Belmonte et al., 1991; MacIver and Tanelian, 1993a, b). Cold receptors are spontaneously active, slowly adapting C-fibres that respond with increased frequency of discharge to cooling of the corneal surface (Lele and Weddell, 1959; Dawson, 1962; Mark and Maurice, 1977; Tanelian and Beuerman, 1984; MacIver and Tanelian, 1993a, b) and which may be of special importance in signalling evaporative cooling of the cornea and initiation of the blink reflex (MacIver and Tanelian, 1993a, b). Whether the cold receptors reported in these studies are truly unimodal has been questioned since in the majority of cases their responses to other modalities of stimulation were not specifically tested; thus, some of them may actually be bimodal “cold nociceptors” (Gallar et al., 1993, see below). Pure chemosensory fibres (i.e., sensory units that are insensitive to mechanical or thermal stimuli) have been described in the rabbit cornea (Tanelian, 1991; MacIver and Tanelian, 1993a, b). The latter fibres are excited by a variety of chemical substances, including, acetylcholine (ACh), nicotine, carbachol, glutamate and its agonist N-methyl-D-aspartate (NMDA), prostaglandin E1, and bradykinin (MacIver and Tanelian, 1993a, b). Similar “pure” chemosensory units have not been identified in the cat, and their existence in the rabbit eye has been questioned on methodological grounds (Belmonte and Gallar, 1996).

Polymodal units are A-δ and C-fibres that respond to all three modalities of stimulation, i.e., to mechanical stimulation, heating of the cornea over 39°C, and select forms of chemical stimulation (e.g., acetic acid, hypertonic saline, and capsaicin) (Belmonte et al., 1991). In contrast, cold is ineffective in exciting these fibres. Sensitivity to protons and to mechanical stimuli in the polymodal fibres may be subserved by separate transduction mechanisms (Belmonte et al., 1991). Bimodal “mechanoheat nociceptor” A-δ fibres have been described by several investigators. Strictly speaking, the latter fibres respond to high threshold mechanosensitive stimuli and to heat, but not to chemical stimuli (Lele and Weddell, 1959; Belmonte and Giraldez, 1981; Belmonte et al., 1991; MacIver and Tanelian, 1993a, b). It has been hypothesized that the latter fibres may actually be polymodal nociceptors, but with atypically high thresholds of activation to chemical stimuli (Belmonte and Gallar, 1996). Finally, cold nociceptors are bimodal units that respond to thermal and chemical stimulation, but not to mechanical stimulation (Gallar et al., 1993).

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Attempts to correlate the electrophysiological properties of corneal nerve fibres with specific epithelial terminal morphologies have been reported by MacIver and Tanelian (1993a, b). These authors stained morphologically distinct subpopulations of living free nerve endings in rabbit corneal epithelium with vital fluorescent dyes and then characterized them electrophysiologically, in vitro. According to their findings, A-δ directionally-sensitive mechanoreceptive endings comprise small clusters of elongated, wavy, thin axons that run horizontally and roughly parallel to one another for long distances. Morphologically, they resemble basal epithelial leashes, but are located more superficially (about 10–40 µm from the corneal surface). In contrast, C-fibre endings are identified by electrical stimulation and form clusters of short (less than 50 µm) poorly branched, vertical, tree-like endings.

TROPHIC FUNCTIONS OF CORNEAL SENSORY NERVES

In addition to their well known sensory functions, corneal afferent nerves exert important nutritive, or trophic, influences on their target organ that contribute to the survival and normal function of the tissue. Damage to the ocular sensory nerves by surgery, trauma, herpes simplex infection or disease produces a degenerative condition in the cornea known as neuroparalytic, or neurotrophic, keratitis (Paton, 1926; Pannabecker, 1944; Davies, 1970). This trophic influence of the trigeminal nerve, first described in rabbit corneas over 170 years ago (Magendie, 1824) has since been demonstrated in numerous mammals following ophthalmic nerve transection or trigeminal ganglion electrocoagulation (Zander and Weddell, 1951b; Rodger, 1953; Sigelman and Friedenwald, 1954; Moses and Feldman, 1969; Alper, 1975; Huhtala, Johansson and Saari, 1975; Lim, 1976; Schimmelpfennig and Beuerman, 1982; Knyazev, Knyazeva and Nikiforov, 1990; Knyazev, Knyazeva and Tolochko, 1991). Keratitis also develops in rats and mice following partial destruction of their corneal innervation by neonatal administration of capsaicin (Figure 2.14) (Keen et al., 1982; Buck et al., 1983; Fujita et al., 1984; Shimizu et al., 1984; Herbort, Weissman and Payan, 1989; Abelli, Geppetti and Maggi, 1993). In capsaicin-treated animals, the severity of the keratitis diminishes in intensity when the nerves reinnervate the cornea (Ogilvy and Borges, 1990; Ogilvy, Silverberg and Borges, 1991; Marfurt, Ellis and Jones, 1993).

Clinically, the development of corneal disturbances in neuroparalytic keratitis follows a characteristic pattern (Paton, 1926; Duke-Elder and Leigh, 1965). Within 24–36 h after nerve injury, the corneal surface becomes stippled and hazy due to progressive epithelial cell death and surface sloughing. Multiple punctate epithelial defects that stain with fluorescein appear first in the centre of the cornea, coalesce into larger areas of cell loss, and rapidly spread into more peripheral areas. If left untreated, massive exfoliation of the epithelium occurs, leaving only a thin, perilimbal ring of intact epithelium. Eventually, the denuded stromal surface becomes dry, milky, and hazy, and the entire cornea may become opaque (edematous) with significantly impaired vision. If secondary infection sets in, corneal ulceration and perforation may follow.

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Studies in experimental animals have contributed significantly to our understanding of the morphological and pathophysiological events that take place in neuroparalytic keratitis. Deprived of their normal sensory innervation, corneal epithelial cells become abnormally rounded, exhibit decreased numbers of surface membrane interdigitations, and a loss of tonofilaments (Sugiura, Kawanabe and Kotashima, 1964; Sugiura and Matsuda, 1967; Lim, 1976; Beuerman, Schimmelpfennig and Burstein, 1979). Superficial epithelial cells become swollen (Gilbard and Rossi, 1990; Knyazev, Knyazeva and Nikiforov, 1990; Knyazev, Knyazeva and Tolochko, 1991) and their surface microvilli are lost or become truncated in appearance (Beuerman, Schimmelpfennig and Burstein, 1979; Gilbard and Rossi, 1990). Intercellular spaces become dilated and epithelial permeability to topical fluorescein increases (Beuerman and Schimmelpfennig, 1980). Total epithelial thickness, cell number, mitotic rate, and epithelial glycogen content are all decreased (Sigelman and Friedenwald, 1954; Mishima, 1957; Alper, 1975; Mackie, 1978; Holden et al., 1982; Beuerman, Tanelian, and Schimmelpfennig, 1988; Gilbard and Rossi, 1990). Massive numbers of leukocytes infiltrate the epithelium and stroma (Knyazev, Knyazeva and Tolochko, 1991). Finally, corneal epithelial oxygen uptake rates are decreased and hypoxic swelling responses are altered (Holden et al., 1982; Vannas et al., 1985).

Corneas deprived of their normal sensory innervation also demonstrate an impaired ability to heal after corneal injuries. Surgical destruction of the rabbit trigeminal ganglion inhibits the rate at which corneal epithelial cells resurface standardized epithelial abrasions (Figures 2.15 and 2.16) (Beuerman and Schimmelpfennig, 1980; Schimmelpfennig and Beuerman, 1982; Araki et al., 1994). The slowed rate of reepithelialization may be related, in part, to the loss of essential cytoskeletal structures necessary for optimal cell migration and adhesion (Beuerman, Schimmelpfennig and Burstein, 1979). Wounded corneas in sensory-denervated eyes also exhibit high numbers of exfoliating surface cells, abnormal migratory cell orientations, and recurrent, spontaneous epithelial erosions (Araki et al., 1994).

Many theories have been proposed to explain the pathogenesis of neuroparalytic keratitis, including, desiccation of the corneal surface due to diminished lacrimal secretions, loss of corneal sensation leading to an absence of normal protective (blink) reflexes, abnormal epithelial cell metabolism with subsequent failure to resist the effects of trauma, drying, and infection, and the loss of trophic impulses supplied by corneal sensory nerves (Paton, 1926; De Haas, 1962; Duke-Elder and Leigh, 1965). Many authors feel that the actual cause is probably represented by a combination of these factors.

A trophic role for corneal nerves is supported by several observations. For example, studies in vivo have shown that corneal abnormalities develop in denervated eyes even if preventative measures are taken to prevent desiccation and trauma by suturing the eyelids (Alper, 1975; Huhtala, Johansson and Saari, 1975; Shimizu et al., 1984; Ogilvy and Borges, 1990). Co-culture of trigeminal ganglion neurons with rabbit corneal epithelial cells stimulates epithelial cell growth, proliferation and differentiation (GarciaHirschfeld, Lopez-Briones and Belmonte, 1994), and epithelial production of type VII collagen (Baker et al., 1993), an important determinant of cell-basement membrane adhesion.

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The mechanisms by which corneal sensory nerves exert their trophic functions are unknown; however, the release of axonally transported substances, particularly neuropeptides, has been postulated. In support of this hypothesis, retrobulbar injection of capsaicin results in depletion of neuropeptides in the rat eye and produces a neuroparalytic keratitis indistinguishable from that seen after total ophthalmic nerve transection (Knyazev, Knyazeva and Nikiforov, 1990). It is tempting to speculate that SP and CGRP subserve ongoing trophic and regulatory processes in the corneal epithelium, and that when released from corneal sensory nerves (either tonically or in response to corneal wounding) stimulate corneal epithelial cells as part of the normal process of tissue maintenance and physiological renewal.

SP and CGRP receptors have been demonstrated on corneal and limbal epithelial cells by autoradiographic analyses (Kieselbach et al., 1990; Denis et al., 1991; Heino et al.,

1995). Cultured rabbit corneal epithelial cells possess approximately 2.43×104 SP binding sites per cell, nearly all of which are of the NK1 subtype (Nakamura et al.,

1997b). Substance P stimulates cell growth and proliferation in primary cultures of rabbit corneal epithelial cells grown in media supplemented with growth factors and hormones (Garcia-Hirschfeld, Lopez-Briones and Belmonte, 1994) and in established rabbit corneal epithelial cells (SIRC cells) grown in a serum-free medium (Reid et al., 1993). In contrast, addition of CGRP alone to either of the above cell lines has no significant effect on DNA synthesis. Of interest, when SP and CGRP are added concurrently to the culture medium, they exert synergistic effects on cell proliferation in the SIRC cells (Reid et al., 1993), but antagonistic effects on proliferation in the primary cell culture (GarciaHirschfeld, Lopez-Briones and Belmonte, 1994) (Figure 2.17).

Recent evidence suggests that SP released from corneal sensory nerve fibres may also promote epithelial cell migration during corneal wound healing. Systemic and topical administration of capsaicin in rabbits depletes ocular neuropeptide levels and impairs the healing rate of standardized corneal epithelial abrasions (Gallar et al., 1990; Murphy et al., 1990). Topical application of SP alone fails to stimulate wound healing in injured rabbit corneas (Nishida et al., 1996; Kingsley and Marfurt, 1997; Nakamura et al., 1997a, b); however, when SP and insulin-like growth factor-1 (IGF-1; Nishida et al., 1996; Nakamura et al., 1997a, b) or SP and epidermal growth factor (Nakamura et al., 1997c) are added together, the neuropeptide and humoral factor act synergistically to stimulate cell migration in a dose-dependent fashion. This effect is mediated by NK1 but not NK2 or NK3, tachykinin receptors (Nakamura et al., 1997b). SP and IGF-1 also act synergistically to promote epithelial cell attachment to extracellular matrix proteins (Nishida et al., 1996). Of clinical interest, topical applications of SP and IGF-1 may promote re-epithelialization in patients with severe neurotrophic and anhidrotic keratopathy (Brown et al., 1997).

The ability of CGRP to stimulate corneal epithelial cell migration is also currently under investigation in several laboratories; however, at the time of this writing the results are inconsistent and vary according to the model of cell migration employed. For example, CGRP has been reported to stimulate migration of rabbit corneal epithelial cells in a whole-cornea organ culture system (Mikulec and Tanelian, 1996), and of SV-40 transformed human corneal epithelial cells in blindwell (chemotaxis) chambers (Lee et al., 1996). In contrast, CGRP has no effect on epithelial cell migration when added to

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cultured rabbit corneal blocks (Nakamura et al., 1997c) or when applied topically to n- heptanol wounded rabbit corneas in vivo (Kingsley and Marfurt, unpublished observations).

Figure 2.17 Effects of application of substance P (SP), calcitonin gene-related peptide (CGRP) and a combination of both, on rabbit corneal epithelial cell proliferation, in vitro. The neuropeptides (0.1 µM–1.0 µm) were applied to cultures during 12-hour or 24-hour experiments. Cell number is expressed as a percentage of control (untreated) values. Results are mean ±S.E.M. of data, *indicates P<0.02. (Reprinted from Garcia-Hirschfeld, Lopez-Briones and Belmonte, 1994, with permission of Academic Press Ltd.).

“OTHER” FUNCTIONS OF CORNEAL SENSORY NEUROPEPTIDES

The extraordinary density of corneal SP-immunoreactive nerves, coupled with the high nociceptive sensibility of the cornea, suggested to early investigators a possible role for SP in peripheral nociceptive transduction mechanisms; however, this hypothesis is now considered unlikely. Topical application of SP antagonists, or depletion of ocular SP levels by capsaicin administration or by herpes simplex virus inoculation, do not eliminate corneal sensitivity or the blink reflex response (Holmdahl et al., 1981; Lembeck and Donnerer, 1981; Keen et al., 1982; Tullo et al., 1983; Bynke, Håkanson

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and Sundler, 1984). These findings demonstrate that impulse transmission in the peripheral parts of corneal SP fibres proceeds without involvement of SP, or, alternatively, that the sensory fibres responsible for corneal nociception are distinct from those containing SP.

In addition to their important roles in corneal trophism and wound healing, SP and CGRP contribute to protective local tissue responses to noxious stimulation of the eye. When the cornea is injured, corneal nerves are stimulated and SP and CGRP are released from anterior uveal nerve fibres by axon-reflex mechanisms. The resulting neurogenic inflammatory response, or “ocular response to injury”, consists of miosis, vasodilation, breakdown of the blood-aqueous barrier, protein extravasation, and a transient elevation in intraocular pressure. The physiological actions of SP and CGRP in this response are subject to considerable interspecies differences and may involve additional mediators such as prostaglandins; however, in most species SP mediates the miotic response and CGRP the vascular reactions leading to raised intraocular pressure and breakdown of the blood aqueous barrier (for reviews see Waldrep, 1989; Unger, 1990; Bill, 1991).

CORNEAL SYMPATHETIC INNERVATION: FUNCTIONAL

CONSIDERATIONS

Corneal sympathetic nerves, like corneal sensory nerves, help promote the anatomical and physiological barrier functions of the corneal epithelium. Studies of corneal sympathetic nerves in animal models have revealed at least four important functions: (i) modulation of epithelial ion transport processes; (ii) regulation of epithelial cell proliferation and mitosis; (iii) modulation of cell migration during epithelial wound healing; (iv) interactions with sensory fibres to exert trophic influences on the cornea.

MODULATION OF EPITHELIAL ION TRANSPORT

Corneal epithelial cells express large numbers of α- and β-adrenoceptors on their cell membranes (Candia and Neufeld, 1978; Neufeld et al., 1978; Cavanagh and Colley, 1982; Walkenbach et al., 1985; Elena et al., 1987, 1990; Walkenbach et al., 1991) and activation of these receptors has been linked to a variety of intracellular processes. In

isolated frog and rabbit corneas, catecholamines stimulate epithelial Cl− transport. According to the model of Klyce and coworkers (Klyce, Beuerman and Crosson, 1985; Klyce and Crosson, 1985) (Figure 2.18), noradrenaline released from rabbit corneal sympathetic nerve fibres activates β-adrenoceptors and results in the stimulation of adenylate cyclase, enhanced production of intracellular adenosine-3′5′-cyclic monophosphate (cAMP), and activation of cAMP-dependent protein kinases (Klyce, Neufeld and Zadunaisky, 1973; Walkenbach and LeGrand, 1981; Walkenbach, LeGrand and Barr, 1981; Reinach and Kirchberger, 1983). These cAMP-dependent protein kinases, in turn, catalyze protein phosphorylation and induce conformational changes in ion channels, leading to increased Cl− permeability of the surface epithelial membranes and a net Cl− transport from stroma to tears (Chalfie,

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Figure 2.18 Proposed model for the mechanism by which various biogenic amines stimulate chloride ion transport across the rabbit corneal epithelium. The scheme is consistent with experimental observations that both sympathectomy and timolol treatment block epithelial responsiveness to serotonin and dopamine. The probable source of serotonin or dopamine is the sympathetic fibres that innervate the cornea. Abbreviations: EPN=adrenaline (epinephrine), NEP=noradrenaline (norepinephrine), TIM=timolol, SER=serotonin, MSD=methysergide, DA=dopamine, HAL=haloperidol, β=β-adrenoceptor, S=serotonin receptor, D=dopamine receptor, AC=adenylate cyclase. (Reprinted from Klyce and Crosson, 1985, with permission of Oxford University Press).

Neufeld and Zadunaisky, 1972; Klyce, Neufeld and Zadunaisky, 1973; Montoreano, Candia and Cook, 1976; Klyce and Wong, 1977; Fischer et al., 1978; Wiederholt et al., 1983). Additional effects on ion transport may be mediated by α1-adrenoceptor stimulation and the hydrolysis of phosphatidylinositol into the second messenger molecules 1,2-diacylglycerol and inositol 1,4,5-triphosphate (Akhtar, 1987).

Additional work in the isolated rabbit corneal model has demonstrated that serotonin

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(Klyce et al., 1982; Jumblatt and Neufeld, 1983; Neufeld, Ledgard and Yoza, 1983; Marshall and Klyce, 1984; Neufeld et al., 1984) and dopamine (Crosson, Beuerman and

Klyce, 1984) also regulate epithelial Cl− transport, most likely via indirect, presynaptic mechanisms. According to Klyce and coworkers (Klyce and Crosson, 1985), serotonin and dopamine released from corneal sympathetic nerve fibres bind to corresponding receptors located on the preterminal portions of the same, or neighbouring, sympathetic nerve fibres. Receptor-mediated events then produce stimulation of chloride ion transport by facilitating the release of noradrenaline.

In theory, it is tempting to speculate that sympathetic stimulation of corneal epithelial chloride transport is physiologically relevant and assists the corneal endothelium in the moment-to-moment control of stromal hydration and corneal transparency. Indeed, work in amphibian corneas suggests a positive correlation between epithelial chloride ion transport and corneal transparency (Zadunaisky and Lande, 1971; Beitsch, Beitsch and Zadunaisky, 1974). In mammals, however, the ion transport capability of the corneal epithelium under normal conditions is only 3–4% that of the corneal endothelium (Klyce, 1982). Clinically, drugs with α- or β-adrenergic activity do not produce corneal stromal hydration problems in patients being treated for primary open-angle glaucoma (Bartels, 1994). Nevertheless, the fact that the corneal epithelium is sympathetically innervated suggests that it may assume some heightened importance during conditions of ocular stress (Klyce and Crosson, 1985). Indeed, patients with unilateral Horner’s Syndrome often present with increased corneal stromal or epithelial thickness in the sympathetically denervated eye (Neilson, 1983; Sweeney et al., 1985) and are less able to control fluid accumulation during hypoxia (Sweeney et al., 1985).

REGULATION OF EPITHELIAL CELL PROLIFERATION

Sympathetic nerves exert pronounced regulatory effects on corneal epithelial cell proliferation (DNA synthesis) and mitosis; however, whether the effects are stimulatory or inhibitory has been the subject of much contention. On the one hand, co-culture of SCG neurons with rabbit corneal epithelial cells increases epithelial cell proliferation by 250% (Garcia-Hirschfeld, Lopez-Briones and Belmonte, 1994). Similarly, ocular sympathetic denervation decreases corneal epithelial proliferation by 30–50% (Figure 2.19) (Jones and Marfurt, 1996) and decreases epithelial mitotic index and mitotic rate by 25–70% (Friedenwald and Buschke, 1944a; Mishima, 1957; Voaden, 1971). On the basis of these findings, it is tempting to speculate that ocular sympathetic nerves release trophic substances (including, noradrenaline) that stimulate corneal epithelial proliferation, and that the decrease in epithelial mitotic activity seen following chronic sympathetic denervation is due to the loss of these substances. In support of this hypothesis, noradrenaline stimulates epithelial cell proliferation in sympathetically-denervated rat eyes in vivo (Jones and Marfurt, 1996) and in human SV-40 transformed epithelial cells in vitro (Murphy et al., 1998).

In contrast, the results of other studies support an inhibitory role for sympathetic innervation on corneal epithelial proliferation and mitosis. Addition of noradrenaline to rabbit corneal epithelial cells in vitro inhibits tritiated thymidine incorporation (Cavanagh and Colley, 1982). Topical adrenaline administration (Friedenwald and Buschke, 1944a)

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and electrical stimulation of the cervical sympathetic trunk (Mishima, 1957) inhibit epithelial mitotic rate in rat and rabbit corneal epithelium, respectively. Extirpation of the rabbit superior cervical ganglion is followed by a sharp decrease in mitotic activity 19–20 hours postoperatively (Mishima, 1957; Butter-field and Neufeld, 1977). This decrease has been hypothesized to be caused by the massive release of noradrenaline from degenerating ocular nerve terminals (Fogle and Neufeld, 1979). The reason for the inconsistent nature of past results in this area remains unknown; however, methodological differences, species differences, and the tendency to equate proliferation with mitosis, may all contribute.

Figure 2.19 The effect of ocular sympathectomy on the labelling index (LI) of the rat corneal epithelium. Four, 14 and 42 days after unilateral removal of the superior cervical ganglion, there is a significant decrease in the ipsilateral epithelial LI. Labelling indices were determined by bromodeoxyuridine (BrdU) incorporation and immunohistochemistry. Values are means ±S.E.M. *Indicates P<0.01. (Reprinted from Jones and Marfurt, 1996, with permission of Lippincott-Raven Publishers.)

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EPITHELIAL WOUND HEALING

When the corneal epithelium is injured, intact cells at the wound margin flatten out and migrate in sheet-like fashion over the denuded area to resurface the defect. Factors which influence the rate of cell migration are multiple and complex and cannot be adequately reviewed here; however, there is increasing evidence that the sympathetic nervous system may play a role in this process. Corneal epithelial wound healing in rabbits is inhibited by sympathetic stimulation (Perez et al., 1987), and accelerated by sympathetic denervation (Beuerman et al., 1985). These data suggest that sympathetic nerves release some substance (possibly noradrenaline) which inhibits the rate of cell migration at the wound margin. In support of this theory, systemic ephedrine or adrenaline inhibit postinjury cell movements in injured rat corneas (Friedenwald and Buschke, 1944b) and topical adrenaline and β-adrenergic blocking agents decrease and increase, respectively, the rate of re-epithelialization in wounded rabbit corneas (Krejci and Harrison, 1970; Reidy et al., 1994). In marked contrast to these findings, other workers have concluded that catecholamines either have no effect (Jumblatt and Neufeld, 1981) or facilitate (Liu, Basu and Trope, 1987; Trope, Liu and Basu, 1988; Liu, Trope and Basu, 1990) the rate of reepithelialization. NE over a wide range of concentrations has also been reported to stimulate migration of transformed human corneal epithelial cells through membrane filters in blindwell chambers (Murphy et al., 1998).

TROPHIC FUNCTIONS

Whether sympathetic nerves exert trophic effects on the cornea is uncertain. Removal of the SCG in experimental animals is apparently without acute, macroscopic effects on corneal transparency or epithelial integrity (e.g., Zander and Weddell, 1951b; Rodger, 1953; Knyazev, Knyazeva and Tolochko, 1991); however, the matter has not been extensively investigated. Clinically, patients with unilateral Horner’s syndrome exhibit only minor abnormalities of corneal physiology. When subjected to a hypoxic “Stress Test”, sympathetically-denervated human eyes show significantly more epithelial graying and microcytic edema, and diminished rates of deswelling (Sweeney et al., 1985).

A small, but intriguing body of literature suggests that maintenance of normal corneal integrity depends on a critical balance of neuronal activity between ocular sympathetic and sensory nerves. For example, the development of neuroparalytic keratitis following trigeminal nerve injury may be prevented or alleviated by removing the superior cervical ganglion (Spalitta, 1894; cited in Paton, 1926) or by cutting the preganglionic inputs to the SCG via stellate ganglionectomy (Harris, 1940; Baker and Gottlieb, 1959). In animal studies, the development of capsaicin-induced, neurotrophic corneal lesions in neonatal rats is prevented or inhibited if the ocular sympathetic nerves are also destroyed (Shimizu et al., 1987; Abelli, Geppetti and Maggi, 1993).

MODULATION OF CORNEAL SENSITIVITY

There has been some speculation that corneal sympathetic fibres modulate the sensitivity

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of the cornea (Laties and Jacobowitz, 1966), possibly by influencing the release of SP from sensory nerve terminals (Morgan, DeGroat and Janetta, 1987). At present, there is little experimental evidence to support this interesting hypothesis. Bernard (1851) observed that extirpation of the cat SCG appears to leave the animals with hypersensitivity of the skin and cornea; however, patients with Horner’s syndrome show no difference in corneal touch threshold between the normal and the sympathetically denervated eye (Sweeney et al., 1985). Studies on peripheral pain mechanisms in other systems suggest that sympathetic nerve stimulation has no effect on the excitability of healthy, primary afferent nociceptors (Shea and Perl, 1985).

THE CORNEAL CHOLINERGIC SYSTEM

The mammalian corneal epithelium contains all of the necessary components of a functional cholinergic system (van Alphen, 1957; Petersen, Keat-Jin and Donn, 1965; Williams and Cooper, 1965; Howard, Wilson and Dunn, 1973; Mindel and Mittag, 1976), including, high levels of ACh, its synthetic enzyme, choline acetyltransferase (ChAT), its degradative enzyme, AChE, and cholinergic receptors. There are apparently two sources of ACh and its associated enzymes in the epithelium: one dependent on the corneal innervation and

one independent of the innervation. Levels of corneal ACh and its enzymes have been reported to either decrease (von Brücke, Hellauer and Umrath, 1949; Petersen, Keat-Jin and Donn, 1965; Fitzgerald and Cooper, 1971) or stay the same (Mindel and Mittag, 1977) following ocular sensory denervation. A non-neuronal source for part of the corneal cholinergic system is supported by the observation that cultured epithelial cells grown in the absence of nerves continue to express the enzymes ChAT and AChE (Gnädinger, Heiman and Markstein, 1973) and by histological demonstrations of AChE in corneal epithelial cell membranes and intercellular spaces (Figure 2.20) (Howard, Zadunaisky and Dunn, 1975; Tervo, 1976; Tervo and Palkama, 1978b).

Pharmacological and radioligand binding studies have demonstrated the presence of muscarinic (but not nicotinic) cholinergic receptors on intact corneal epithelial cells of rabbits and humans (Cavanagh and Colley, 1982; Colley and Cavanagh, 1982; Walkenbach and Ye, 1991). Work by Cavanagh and coworkers (Colley et al., 1985; Lind and Cavanagh, 1993) suggests that the receptors are expressed not only on the surface of the epithelial cells but also on the epithelial nuclear membrane.

FUNCTIONAL CONSIDERATIONS

A definitive role for the cholinergic system in corneal physiology remains elusive; however, several possible functions have been proposed. Corneal ACh may play a role in corneal epithelial cell growth, proliferation, and the healing of epithelial defects (Figure 2.21). The addition of cholinergic agonists to corneal epithelial cell cultures increases intracellular guanosine-3′5′-cyclic monophosphate (cGMP) production (Cavanagh and Colley, 1982; Colley and Cavanagh, 1982; Walkenbach and Ye, 1991) stimulates [3H]- thymidine and [14C]-leucine incorporation (Cavanagh and Colley, 1982), enhances

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Figure 2.21 A model for cholinergic regulation of epithelial cellular growth. (Reprinted from Lind and Cavanagh, 1993, with permission of Lippincott-Raven Publishers).

ACh has also been implicated in the regulation of corneal epithelial ion transport processes. Depletion of endogenous corneal ACh levels by administration of ChAT inhibitors decreases Na+ and Cl− transport in frog corneal epithelium (Pesin and Candia, 1982) and increases Na+ transport into the corneal stroma of preswollen rabbit corneas (Stevenson and Wilson, 1975).

ACh has also been postulated to play a role in corneal sensory transduction mechanisms. In most mammals, there is a positive correlation between corneal sensitivity and epithelial ACh content. Species that possess high corneal ACh content require less stimulation, using von Frey hairs, to elicit a blink reflex than do species with low ACh content (Hellauer, 1950). Corneal ACh content, ChAT activity and corneal sensitivity are highest in the central corneal epithelium and lowest in the peripheral epithelium (Hellauer, 1950; Mindel and Mittag, 1976, 1977; Wilson and McKean, 1986). Corneal ACh content and corneal sensitivity decrease proportionately in aged individuals, after eyelid suturing, and following administration of anticholinergic drugs (Hellauer, 1950; Fitzgerald and Cooper, 1971; Mindel and Mittag, 1976, 1977; Millodot and O’Leary, 1979). Application of ACh to the corneal surface evokes action potentials in rabbit C- fibre afferents (Tanelian, 1991) and causes eye pain in humans (Jancso, Jancso-Gabor and Takats, 1961). Taken collectively, these data suggest that a certain critical level of ACh is requisite to corneal sensitivity, and that ACh released in the vicinity of corneal nerve terminals following corneal injury may produce pain. It should be cautioned, however, that much of the evidence in support of this hypothesis is indirect, and that a role for ACh in corneal sensory transduction processes is not universally accepted.

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DEVELOPMENT OF THE CORNEAL INNERVATION

Development of the corneal innervation has been described for several mammalian and avian species with similar findings (Kitano, 1957; Lúkas and Dolezel, 1975; Ozanics, Rayborn and Sagun, 1977; Tervo, Tervo and Palkama, 1978; Tervo and Tervo, 1981; Bee, 1982; Bee et al., 1986). The innervation of the embryonic chick cornea has been especially well described by Bee and coworkers (Bee, 1982; Bee et al., 1982, 1986) and can be subdivided into two distinct phases. In Phase I (embryonic days 6–10), the ocular nerves approach (but do not penetrate) the ventrotemporal region of the corneal limbus. The nerves then separate into two groups which extend ventrally and dorsally around the cornea before meeting each other to form a prominent ring-like latticework of encircling nerve fibres (Figure 2.22). The pathway followed by the elongating axons is determined in part by the nerves’ ability to recognize tissue-derived guidance cues (e.g., glycosaminoglycans) in the extracellular matrix (Bee et al., 1982). Soon thereafter, the nerves penetrate the peripheral corneal stroma and begin growing towards the central zone. Over the next several months, the fibres increase in number and branching complexity. Axonal bifurcations occur within distinct concentric zones which are, for unknown reasons, conducive to nerve branching (Bee et al., 1986).

In the human embryo, ciliary nerves reach the optic cup about midway through the first trimester, and first enter the cornea at about 3 months (Kitano, 1957). By 4 months, stromal nerves are present in large numbers beneath Bowman’s membrane, and from 4 to 9 months the nerves undergo a gradual maturation process to reach the adult-like arrangement at time of birth.

Axonal penetration into the corneal epithelium first occurs at about 5 months in humans (Kitano, 1957), a little earlier in monkeys (Ozanics, Rayborn and Sagun, 1977), and apparently not until birth in the rat (Singh and Beuerman, 1979; Tervo and Tervo, 1981). The mechanisms that direct the developing axons into the epithelium are largely unknown;

however, it is known that embryonic and mature epithelial cells produce diffusible neuronotropic “factors” which promote neurite extension (Chan and Haschke, 1982, 1985; Emoto and Beuerman, 1987; Pavlidis, Steuhl and Thanos, 1994). At the electron microscopic level, the first recognizable epithelial nerve terminals contain small numbers of large, dense-cored vesicles, a few mitochondria and, at a slightly later stage, smaller vesicles (Ozanics, Rayborn and Sagun, 1977). The number of vesicles in the nerve terminals then increases progressively as a function of time (Tervo and Tervo, 1981). The mitochondria in the primitive terminals are smaller, more dense, and differ in internal configuration from the mitochondria in surrounding corneal epithelial cells, suggesting high levels of neural metabolic activity (Ozanics, Rayborn and Sagun, 1977).

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Figure 2.22 Initial stage of migration and positioning of corneal nerves in the avian cornea. The micrograph illustrates a whole-mounted cornea from a 7 day-old chick embryo. Numerous large nerve fascicles approach (but do not enter) the cornea and migrate both dorsally (d) and ventrally (v) from their origin (O). Eventually, the nerve groups will meet one another and form a complete ring around the periphery of the cornea. Scale bar=200 µm. (Reprinted from Bee, 1982, with permission of Academic Press, Inc.).

In the rat, corneal nerve development continues for several weeks after birth (Jones and Marfurt, 1991). At the time of birth, the intraepithelial segments of the nerves are morphologically simple and support few intraepithelial terminals. Over the next three weeks, the epithelial segments increase significantly in length, branching complexity, degree of radial orientation, and richness of terminal elements, finally attaining their adult-like morphology on about postnatal day 21 (Figure 2.23).

Neuropeptides are first observed immunohistochemically in the developing rat cornea approximately 3–4 days prior to birth (Sakiyama et al., 1984). SP is seen in developing chick corneal nerves only after the nerves penetrate the epithelium, prompting speculation that SP expression is initiated either by physical contact with the epithelial cells or in response to the elaboration of one or more epithelium-derived neuronotrophic factors (Bee et al., 1988; Corvetti, Pignocchino and Sisto Daneo, 1988).

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elongate and increase in overall complexity; immature versions of adult leash formations are recognizable at about day 15. (Reprinted from Jones and Marfurt, 1991, with permission of John Wiley and Sons, Inc.)

Functionally, it has been suggested that corneal nerves and their neurochemicals may trigger certain aspects of embryonic and postnatal corneal development. For example, dehydration of the chick cornea and the change from corneal opacity to transparency correlates with the arrival of corneal nerves in the stroma (Bee, 1982; Clarke and Bee, 1996). SP-IR nerves in the chick corneal epithelium increase in number and branching complexity between embryonic days 12 and 17 (Bee et al., 1988; Corvetti, Pignocchino and Sisto Daneo, 1988); at the same time, the corneal epithelium begins to differentiate (Hay, 1979). Whether the latter events are causal or coincidental remains to be shown. It has been hypothesized that the acceleration of corneal epithelial differentiation seen postnatally may be due to enhanced neurotransmitter release from sensory nerve terminals as the result of repeated blinking of the newly opened lids (Watanabe, Tisdale and Gipson, 1993). Neonatal administration of capsaicin in mice and rats significantly depletes the normal SP and CGRP content of the developing corneas; several weeks later these corneas develop epithelial abnormalities and chronic neuroparalytic keratitis (Fujita et al., 1984; Marfurt, Ellis and Jones, 1993). In contrast to the situation in chick and rat eyes, differentiation in the rabbit embryonic cornea clearly begins prior to the invasion of nerve fibres (Lúkas and Dolezel, 1975).

The development of the corneal sympathetic innervation has been described by several investigators on the basis of histochemical fluorescence and electron microscopic observations. Fluorescent adrenergic nerves are first observed in the developing human cornea in the second trimester and in the rat, guinea pig and rabbit cornea near the beginning of the third trimester (Ehinger and Sjöberg, 1971; Lúkas and Dolezel, 1975; Tervo, Tervo and Palkama, 1978). Morphologically, embryonic adrenergic nerves are smoother than their adult counterparts and lack varicosities (Ehinger and Sjöberg, 1971; Tervo, 1977), suggesting a certain level of functional immaturity. The number of stromal and epithelial fibres, and the intensity with which the nerves fluoresce, increase progressively throughout development, reaching a maximum shortly before, or immediately after, birth (Ehinger, 1966c; Ehinger and Sjöberg, 1971; Tervo, 1977; Tervo, Tervo and Palkama, 1978; Toivanen et al., 1987). The numbers of adrenergic fibres then decline progressively, even precipitously, in the weeks immediately following birth to quickly reach adult levels. Most of this fibre loss is probably due to extensive postnatal cell death in the neonatal SCG (Vidovic and Hill, 1988), due to an inability of the sympathetic nerves to successfully compete with the corneal sensory nerve fibres for available growth factors. Alternatively, postnatal increases in corneal size (Tervo, Tervo and Palkama, 1978) or accelerated rates of neurotransmitter release by mature nerve fibres may also explain why adult corneas appear to contain fewer nerves than fetal corneas.

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ACKNOWLEDGEMENTS

The author expresses his deepest appreciation to Drs. Carlos Belmonte, Mark Jones, Robert Kingsley, and Gordon Ruskell for their invaluable comments on earlier versions of this manuscript. I would also like to thank the following individuals for generously providing original photomicrographs of their work for use in this paper: Kaoru ArakiSasaki, Carlos Belmonte, Roger Beuerman, Juana Gallar, Carl Herbort, Mark Jones, Linda Müller, and Timo Tervo. This work was supported by NIH grant EY05717.

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